Release of insulin granules by simultaneous, high-speed correlative SICM-FCM.

Exocytosis of peptides and steroids stored in a dense core vesicular (DCV) form is the final step of every secretory pathway, indispensable for the function of nervous, endocrine and immune systems. The lack of live imaging techniques capable of direct, label-free visualisation of DCV release makes many aspects of the exocytotic process inaccessible to investigation. We describe the application of correlative scanning ion conductance and fluorescence confocal microscopy (SICM-FCM) to study the exocytosis of individual granules of insulin from the top, nonadherent, surface of pancreatic ß-cells. Using SICM-FCM, we were first to directly follow the topographical changes associated with physiologically induced release of insulin DCVs. This allowed us to report the kinetics of the full fusion of the insulin vesicle as well as the subsequent solubilisation of the released insulin crystal.

Incretin hormones, insulin, glucagon and advanced glycation end products in relation to cognitive function in older people with and without diabetes, a population-based study.

AIM: The aim of this observational study was to investigate relationships between physiological levels of glucometabolic biomarkers and cognitive test results in a population-based setting. METHODS: Cross-sectional data were obtained from the Swedish population-based Malmö Diet and Cancer Study Re-examination 2007-2012 comprising 3001 older people (mean age 72 years). Through oral glucose tolerance testing (OGTT), fasting and post-load levels of serum insulin, plasma glucagon, serum glucose-dependent insulinotropic peptide (GIP) and plasma glucagon-like peptide-1 (GLP-1) were measured. Insulin resistance and insulin sensitivity levels were calculated. In 454 participants, advanced glycation end products (AGEs) were estimated through skin autofluorescence. Associations between biomarkers and two cognitive tests, the Mini-Mental State Examination (MMSE) and A Quick Test of Cognitive Speed (AQT) respectively, were explored in multiple regression analyses. RESULTS: Positive associations following adjustments for known prognostic factors were found between MMSE scores and insulin sensitivity (B = 0.822, P = 0.004), 2-h plasma glucagon (B = 0.596, P = 0.026), 2-h serum GIP (B = 0.581, P = 0.040) and 2-h plasma GLP-1 (B = 0.585, P = 0.038), whereas negative associations were found between MMSE scores and insulin resistance (B = -0.734, P = 0.006), fasting plasma GLP-1 (B = -0.544, P = 0.033) and AGEs (B = -1.459, P = 0.030) were found. CONCLUSIONS: Higher levels of insulin sensitivity, GIP and GLP-1 were associated with better cognitive outcomes, but AGEs were associated with worse outcomes, supporting evidence from preclinical studies. Glucagon was linked to better outcomes, which could possibly reflect neuroprotective properties similar to the related biomarker GLP-1 which has similar intracellular properties. Longitudinal and interventional studies are needed to further evaluate neuromodulating effects of these biomarkers. Abstract presented at the European Association for the Study of Diabetes (EASD) 2019, Barcelona, Spain.

δ-cells and ß-cells are electrically coupled and regulate α-cell activity via somatostatin.

KEY POINTS: We used a mouse expressing a light-sensitive ion channel in ß-cells to understand how α-cell activity is regulated by ß-cells. Light activation of ß-cells triggered a suppression of α-cell activity via gap junction-dependent activation of δ-cells. Mathematical modelling of human islets suggests that 23% of the inhibitory effect of glucose on glucagon secretion is mediated by ß-cells via gap junction-dependent activation of δ-cells/somatostatin secretion. ABSTRACT: Glucagon, the body's principal hyperglycaemic hormone, is released from α-cells of the pancreatic islet. Secretion of this hormone is dysregulated in type 2 diabetes mellitus but the mechanisms controlling secretion are not well understood. Regulation of glucagon secretion by factors secreted by neighbouring ß- and δ-cells (paracrine regulation) have been proposed to be important. In this study, we explored the importance of paracrine regulation by using an optogenetic strategy. Specific light-induced activation of ß-cells in mouse islets expressing the light-gated channelrhodopsin-2 resulted in stimulation of electrical activity in δ-cells but suppression of α-cell activity. Activation of the δ-cells was rapid and sensitive to the gap junction inhibitor carbenoxolone, whereas the effect on electrical activity in α-cells was blocked by CYN 154806, an antagonist of the somatostatin-2 receptor. These observations indicate that optogenetic activation of the ß-cells propagates to the δ-cells via gap junctions, and the consequential stimulation of somatostatin secretion inhibits α-cell electrical activity by a paracrine mechanism. To explore whether this pathway is important for regulating α-cell activity and glucagon secretion in human islets, we constructed computational models of human islets. These models had detailed architectures based on human islets and consisted of a collection of >500 α-, ß- and δ-cells. Simulations of these models revealed that this gap junctional/paracrine mechanism accounts for up to 23% of the suppression of glucagon secretion by high glucose.

Key Matrix Proteins Within the Pancreatic Islet Basement Membrane Are Differentially Digested During Human Islet Isolation.

Clinical islet transplantation achieves insulin independence in selected patients, yet current methods for extracting islets from their surrounding pancreatic matrix are suboptimal. The islet basement membrane (BM) influences islet function and survival and is a critical marker of islet integrity following rodent islet isolation. No studies have investigated the impact of islet isolation on BM integrity in human islets, which have a unique duplex structure. To address this, samples were taken from 27 clinical human islet isolations (donor age 41-59, BMI 26-38, cold ischemic time < 10 h). Collagen IV, pan-laminin, perlecan and laminin-α5 in the islet BM were significantly digested by enzyme treatment. In isolated islets, laminin-α5 (found in both layers of the duplex BM) and perlecan were lost entirely, with no restoration evident during culture. Collagen IV and pan-laminin were present in the disorganized BM of isolated islets, yet a significant reduction in pan-laminin was seen during the initial 24 h culture period. Islet cytotoxicity increased during culture. Therefore, the human islet BM is substantially disrupted during the islet isolation procedure. Islet function and survival may be compromised as a consequence of an incomplete islet BM, which has implications for islet survival and transplanted graft longevity.

Multivesicular exocytosis in rat pancreatic beta cells.

AIMS/HYPOTHESIS: To establish the occurrence, modulation and functional significance of compound exocytosis in insulin-secreting beta cells. METHODS: Exocytosis was monitored in rat beta cells by electrophysiological, biochemical and optical methods. The functional assays were complemented by three-dimensional reconstruction of confocal imaging, transmission and block face scanning electron microscopy to obtain ultrastructural evidence of compound exocytosis. RESULTS: Compound exocytosis contributed marginally (<5% of events) to exocytosis elicited by glucose/membrane depolarisation alone. However, in beta cells stimulated by a combination of glucose and the muscarinic agonist carbachol, 15-20% of the release events were due to multivesicular exocytosis, but the frequency of exocytosis was not affected. The optical measurements suggest that carbachol should stimulate insulin secretion by ∼40%, similar to the observed enhancement of glucose-induced insulin secretion. The effects of carbachol were mimicked by elevating [Ca(2+)](i) from 0.2 to 2 µmol/l Ca(2+). Two-photon sulforhodamine imaging revealed exocytotic events about fivefold larger than single vesicles and that these structures, once formed, could persist for tens of seconds. Cells exposed to carbachol for 30 s contained long (1-2 µm) serpentine-like membrane structures adjacent to the plasma membrane. Three-dimensional electron microscopy confirmed the existence of fused multigranular aggregates within the beta cell, the frequency of which increased about fourfold in response to stimulation with carbachol. CONCLUSIONS/INTERPRETATION: Although contributing marginally to glucose-induced insulin secretion, compound exocytosis becomes quantitatively significant under conditions associated with global elevation of cytoplasmic calcium. These findings suggest that compound exocytosis is a major contributor to the augmentation of glucose-induced insulin secretion by muscarinic receptor activation.

Autocrine regulation of insulin secretion.

Impaired insulin secretion from pancreatic ß-cells is a major factor in the pathogenesis of type 2 diabetes. The main regulator of insulin secretion is the plasma glucose concentration. Insulin secretion is modified by other nutrients, circulating hormones and the autonomic nervous system, as well as local paracrine and autocrine signals. Autocrine signalling involves diffusible molecules that bind to receptors on the same cell from which they have been released. The first transmitter to be implicated in the autocrine regulation of ß-cell function was insulin itself. The importance of autocrine insulin signalling is underscored by the finding that mice lacking insulin receptors in ß-cells are glucose intolerant. In addition to insulin, ß-cells secrete a variety of additional substances, including peptides (e.g. amylin, chromogranin A and B and their cleavage products), neurotransmitters (ATP and γ-aminobutyric acid) and ions (e.g. zinc). Here we review the autocrine effects of substances secreted from ß-cells, with a focus on acute effects in stimulus-secretion coupling, present some novel data and discuss the general significance of autocrine signals for the regulation of insulin secretion.

Per-arnt-sim (PAS) domain kinase (PASK) as a regulator of glucagon secretion.

The physiological and pathophysiological regulation of glucagon secretion from pancreatic alpha cells remains a hotly debated topic. The mechanism(s) contributing to the glucose sensitivity of glucagon release and its impaired regulation in diabetes remain unclear. A paper in the current issue of Diabetologia by da Silva Xavier and colleagues (doi: 10.1007/s00125-010-2010-7 ) provides intriguing new insight into a metabolic sensing pathway mediated by the per-arnt-sim (PAS) domain kinase (PASK) that may contribute to both the paracrine and the intrinsic glucose regulation of alpha cells. Importantly, the authors show that PASK is decreased in islets from patients with type 2 diabetes, providing a potential mechanism for impaired suppression of glucagon by hyperglycaemia in this disease. Much work remains to be done to determine the exact role and mechanism of PASK in alpha and beta cells. Nevertheless, the present work introduces a new player in the metabolic regulation of glucagon secretion.

Regulation of glucagon secretion by glucose: paracrine, intrinsic or both?

Glucagon secretion is regulated by glucose but the mechanisms involved remain hotly debated. Both intrinsic (within the α-cell itself) and paracrine (mediated by factors released ß- and/or δ-cells) have been postulated. Glucagon secretion is maximally suppressed by glucose concentrations that do not affect insulin and somatostatin secretion, a finding that highlights the significance of intrinsic regulation of glucagon secretion. Experiments on islets from mice lacking functional ATP-sensitive potassium channels (K(ATP)-channels) indicate that these channels are critical to the α-cell's capacity to sense changes in extracellular glucose. Here, we review recent data on the intrinsic and paracrine regulation of glucagon secretion in human pancreatic islets. We propose that glucose-induced closure of the K(ATP)-channels, via membrane depolarization, culminates in reduced electrical activity and glucagon secretion by voltage-dependent inactivation of the ion channels involved in action potential firing. We further demonstrate that glucagon secretion measured in islets isolated from donors with type-2 diabetes is reduced at low glucose and that glucose stimulates rather than inhibits secretion in these islets. We finally discuss the relative significance of paracrine and intrinsic regulation in the fed and fasted states and propose a unifying model for the regulation of glucagon secretion that incorporates both modes of control.

The glucagon-producing alpha cell: an electrophysiologically exceptional cell.

Activation of potassium channels normally serves to reduce cellular activity but recent data indicate that the glucagon-secreting alpha cells are different in this respect and that inhibition of voltage-gated potassium channels results in a paradoxical inhibition of glucagon secretion. Here we discuss these findings and attempt to provide a model for the regulation of glucagon secretion that incorporates these observations.

Enhancement of glucagon secretion in mouse and human pancreatic alpha cells by protein kinase C (PKC) involves intracellular trafficking of PKCalpha and PKCdelta.

AIMS/HYPOTHESIS: Protein kinase C (PKC) regulates exocytosis in various secretory cells. Here we studied intracellular translocation of the PKC isoenzymes PKCalpha and PKCdelta, and investigated how activation of PKC influences glucagon secretion in mouse and human pancreatic alpha cells. METHODS: Glucagon release from intact islets was measured in static incubations, and the amounts released were determined by RIA. Exocytosis was monitored as increases in membrane capacitance using the patch-clamp technique. The expression of genes encoding PKC isoforms was analysed by real-time PCR. Intracellular PKC distribution was assessed by confocal microscopy. RESULTS: The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated glucagon secretion from mouse and human islets about fivefold (p < 0.01). This stimulation was abolished by the PKC inhibitor bisindolylmaleimide (BIM). Whereas PMA potentiated exocytosis more than threefold (p < 0.001), BIM inhibited alpha cell exocytosis by 60% (p < 0.05). In mouse islets, the PKC isoenzymes, PKCalpha and PKCbeta1, were highly abundant, while in human islets PKCeta, PKCepsilon and PKCzeta were the dominant variants. PMA stimulation of human alpha cells correlated with the translocation of PKCalpha and PKCdelta from the cytosol to the cell periphery. In the mouse alpha cells, PKCdelta was similarly affected by PMA, whereas PKCalpha was already present at the cell membrane in the absence of PMA. This association of PKCalpha in alpha cells was principally dependent on Ca(2+) influx through the L-type Ca(2+) channel. CONCLUSIONS/INTERPRETATION: PKC activation augments glucagon secretion in mouse and human alpha cells. This effect involves translocation of PKCalpha and PKCdelta to the plasma membrane, culminating in increased Ca(2+)-dependent exocytosis. In addition, we demonstrated that PKCalpha translocation and exocytosis exhibit differential Ca(2+) channel dependence.

Somatostatin release, electrical activity, membrane currents and exocytosis in human pancreatic delta cells.

AIMS/HYPOTHESIS: The aim of this study was to characterise electrical activity, ion channels, exocytosis and somatostatin release in human delta cells/pancreatic islets. METHODS: Glucose-stimulated somatostatin release was measured from intact human islets. Membrane potential, currents and changes in membrane capacitance (reflecting exocytosis) were recorded from individual human delta cells identified by immunocytochemistry. RESULTS: Somatostatin secretion from human islets was stimulated by glucose and tolbutamide and inhibited by diazoxide. Human delta cells generated bursting or sporadic electrical activity, which was enhanced by tolbutamide but unaffected by glucose. Delta cells contained a tolbutamide-insensitive, Ba(2+)-sensitive inwardly rectifying K(+) current and two types of voltage-gated K(+) currents, sensitive to tetraethylammonium/stromatoxin (delayed rectifying, Kv2.1/2.2) and 4-aminopyridine (A current). Voltage-gated tetrodotoxin (TTX)-sensitive Na(+) currents contributed to the action potential upstroke but TTX had no effect on somatostatin release. Delta cells are equipped with Ca(2+) channels blocked by isradipine (L), omega-agatoxin (P/Q) and NNC 55-0396 (T). Blockade of any of these channels interferes with delta cell electrical activity and abolishes glucose-stimulated somatostatin release. Capacitance measurements revealed a slow component of depolarisation-evoked exocytosis sensitive to omega-agatoxin. CONCLUSIONS/INTERPRETATION: Action potential firing in delta cells is modulated by ATP-sensitive K(+)-channel activity. The membrane potential is stabilised by Ba(2+)-sensitive inwardly rectifying K(+) channels. Voltage-gated L- and T-type Ca(2+) channels are required for electrical activity, whereas Na(+) currents and P/Q-type Ca(2+) channels contribute to (but are not necessary for) the upstroke of the action potential. Action potential repolarisation is mediated by A-type and Kv2.1/2.2 K(+) channels. Exocytosis is tightly linked to Ca(2+)-influx via P/Q-type Ca(2+) channels. Glucose stimulation of somatostatin secretion involves both K(ATP) channel-dependent and -independent processes.

Increased activity of L-type Ca2+ channels exposed to serum from patients with type I diabetes.

Type I diabetes [insulin-dependent diabetes mellitus (IDDM)] is an autoimmune disease associated with the destruction of pancreatic beta cells. Serum from patients with IDDM increased L-type calcium channel activity of insulin-producing cells and of GH3 cells derived from a pituitary tumor. The subsequent increase in the concentration of free cytoplasmic Ca2+ ([Ca2+]i) was associated with DNA fragmentation typical of programmed cell death or apoptosis. These effects of the serum were prevented by adding a blocker of voltage-activated L-type Ca2+ channels. When the serum was depleted of immunoglobulin M (IgM), it no longer affected [Ca2+]i. An IgM-mediated increase in Ca2+ influx may thus be part of the autoimmune reaction associated with IDDM and contribute to the destruction of beta cells in vivo.

PKC-dependent stimulation of exocytosis by sulfonylureas in pancreatic beta cells.

Hypoglycemic sulfonylureas represent a group of clinically useful antidiabetic compounds that stimulate insulin secretion from pancreatic beta cells. The molecular mechanisms involved are not fully understood but are believed to involve inhibition of potassium channels sensitive to adenosine triphosphate (KATP channels) in the beta cell membrane, causing membrane depolarization, calcium influx, and activation of the secretory machinery. In addition to these effects, sulfonylureas also promoted exocytosis by direct interaction with the secretory machinery not involving closure of the plasma membrane KATP channels. This effect was dependent on protein kinase C (PKC) and was observed at therapeutic concentrations of sulfonylureas, which suggests that it contributes to their hypoglycemic action in diabetics.

Neurotransmitter-induced inhibition of exocytosis in insulin-secreting beta cells by activation of calcineurin.

Neurotransmitters and hormones such as somatostatin, galanin, and adrenalin reduce insulin secretion. Their inhibitory action involves direct interference with the exocytotic machinery. We have examined the molecular processes underlying this effect using high resolution measurements of cell capacitance. Suppression of exocytosis was maximal at concentrations that did not cause complete inhibition of glucose-stimulated electrical activity. This action was dependent on activation of G proteins but was not associated with inhibition of the voltage-dependent Ca2+ currents or adenylate cyclase activity. The molecular processes initiated by the agonists culminate in the activation of the Ca(2+)-dependent protein phosphatase calcineurin, and suppression of the activity of this enzyme abolishes their action on exocytosis. We propose that mechanisms similar to those we report here may contribute to adrenergic and peptidergic inhibition of secretion in other neuroendocrine cells and in nerve terminals.

Enhanced stimulus-secretion coupling in polyamine-depleted rat insulinoma cells. An effect involving increased cytoplasmic Ca2+, inositol phosphate generation, and phorbol ester sensitivity.

To extend previous observations on the role of polyamines in insulin production, metabolism, and replication of insulin-secreting pancreatic beta cells, we have studied the role of polyamines in the regulation of the stimulus-secretion coupling of clonal rat insulinoma cells (RINm5F). For this purpose, RINm5F cells were partially depleted in their polyamine contents by use of the specific ornithine decarboxylase inhibitor difluoromethylornithine (DFMO), which led to an increase in cellular insulin and ATP contents. Analysis of different parts of the signal transduction pathway revealed that insulin secretion and the increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) after K(+)-induced depolarization were markedly enhanced in DFMO-treated cells. These effects were paralleled by increased voltage-activated Ca2+ currents, as judged by whole-cell patch-clamp analysis, probably reflecting increased channel activity rather than elevated number of channels per cell. DFMO treatment also rendered phospholipase C in these cells more sensitive to the muscarinic receptor agonist carbamylcholine, as evidenced by enhanced generation of inositol phosphates, increase in [Ca2+]i and insulin secretion, despite an unaltered ligand binding to muscarinic receptors and lack of effect on protein kinase C activity. In addition, the tumor promoter 12-O-tetradecanoylphorbol 13-acetate, at concentrations suggested to be specific for protein kinase C activation, evoked an increased insulin output in polyamine-deprived cells compared to control cells. The stimulatory effects of glucose or the cyclic AMP raising agent theophylline on insulin release were not increased by DFMO treatment. In spite of increased binding of sulfonylurea in DFMO-treated cells, there was no secretory response or altered increase in [Ca2+]i in response to the drug in these cells. It is concluded that partial polyamine depletion sensitizes the stimulus-secretion coupling at multiple levels in the insulinoma cells, including increased voltage-dependent Ca2+ influx and enhanced responsiveness to activators of phospholipase C and protein kinase C. In their entirety, our present results indicate that the behavior of the stimulus-secretion coupling of polyamine-depleted RINm5F insulinoma cells changes towards that of native beta cells, thus improving the usefulness of this cell line for studies of beta cell insulin secretion.

Inhibition of ATP-regulated K(+)-channels by a photoactivatable ATP-analogue in mouse pancreatic beta-cells.

The effects of a photoactivable (DMNPE-caged) ATP-analogue on ATP-regulated K(+)-channels (KATP-channel) in mouse pancreatic beta-cells were investigated using the inside-out patch configuration of the patch-clamp technique. The caged precursor caused a concentration-dependent reduction of channel activity with a Ki of 17 microM; similar to the 11 microM obtained for standard Mg-ATP. The small difference in the blocking capacity between the precursor and ATP is probably the reason why no change in channel activity was observed upon photolysis of the caged molecule and liberation of ATP. It is suggested that the part of the ATP molecule involved in the blocking reaction of the KATP-channel is not sufficiently protected in DMNPE-caged ATP making this compound unsuitable for studying the rapid kinetics of ATP-induced KATP-channel inhibition.

Intracellular pH, cytosolic calcium concentration and electrical activity in RINm5F insulinoma cells.

The addition of L-lactate or acetate to RINm5F cells caused a transient intracellular acidification, an increase in [Ca2+]i and induced electrical activity. The subsequent withdrawal of lactate or acetate resulted in an intracellular alkalinization with no apparent changes in [Ca2+]i nor electrical activity. Intracellular alkalinization and acidification by application by application and withdrawal of NH4Cl were both accompanied by transient increases in [Ca2+]i in the absence of electrical activity. The induction of electrical activity by lactate was associated with the appearance of inward whole cell currents. Changes in intracellular pH may affect [Ca2+]i though not necessarily by altering plasma membrane potential. The inward currents associated with lactate application may represent an organic anion conductance contributing towards the stimulation of electrical activity by organic acids.

Tight coupling between electrical activity and exocytosis in mouse glucagon-secreting alpha-cells.

alpha-Cells were identified in preparations of dispersed mouse islets by immunofluorescence microscopy. A high fraction of alpha-cells correlated with a small cell size measured as the average cell diameter (10 microm) and whole-cell capacitance (<4 pF). The alpha-cells generated action potentials at a low frequency (1 Hz) in the absence of glucose. These action potentials were reversibly inhibited by elevation of the glucose concentration to 20 mmol/l. The action potentials originated from a membrane potential more negative than -50 mV, had a maximal upstroke velocity of 5 V/s, and peaked at +1 mV. Voltage-clamp experiments revealed the ionic conductances underlying the generation of action potentials. alpha-Cells are equipped with a delayed tetraethyl-ammonium-blockable outward current (activating at voltages above -20 mV), a large tetrodotoxin-sensitive Na+ current (above -30 mV; peak current 200 pA at +10 mV), and a small Ca2+ current (above -50 mV; peak current 30 pA at +10 mV). The latter flowed through omega-conotoxin GVIA (25%)- and nifedipine-sensitive (50%) Ca(2+)-channels. Mouse alpha-cells contained, on average, 7,300 granules, which undergo Ca(2+)-induced exocytosis when the alpha-cell is depolarized. Three functional subsets of granules were identified, and the size of the immediately releasable pool was estimated as 80 granules, or 1% of the total granule number. The maximal rate of exocytosis (1.5 pF/s) was observed 21 ms after the onset of the voltage-clamp depolarization, which is precisely the duration of Ca(2+)-influx during an action potential. Our results suggest that the secretory machinery of the alpha-cell is optimized for maximal efficiency in the use of Ca2+ for exocytosis.

Glucagon-like peptide I and glucose-dependent insulinotropic polypeptide stimulate Ca2+-induced secretion in rat alpha-cells by a protein kinase A-mediated mechanism.

High-resolution capacitance measurements were used to explore the effects of the gut hormones GLP-I(7-36) amide [glucagon-like peptide I(7-36) amide] and GIP (glucose-dependent insulinotropic polypeptide) on Ca2+-dependent exocytosis in glucagon-secreting rat pancreatic alpha-cells. Both peptides produced a greater than threefold potentiation of secretion evoked by voltage-clamp depolarizations, an effect that was associated with an approximately 35% increase of the Ca2+ current. The stimulatory actions of GLP-I(7-36) amide and GIP were mimicked by forskolin and antagonized by the protein kinase A (PKA)-inhibitor Rp-8-Br-cAMPS. The islet hormone somatostatin inhibited the stimulatory action of GLP-I(7-36) amide and GIP via a cyclic AMP-independent mechanism, whereas insulin had no effect on exocytosis. These data suggest that the alpha-cells are equipped with receptors for GLP-I and GIP and that these peptides, in addition to their well-established insulinotropic capacity, also stimulate glucagon secretion. We propose that the reported inhibitory action of GLP-I on glucagon secretion is accounted for by a paracrine mechanism (e.g., mediated by stimulated release of somatostatin that in turn suppresses exocytosis in the alpha-cell).